The present invention relates generally to hydrated lipidic lamellar phases and to a method for obtaining the same, and also to compositions intended particularly for pharmaceutical, cosmetic or like uses and comprising said lipidic lamellar phases.
The present invention is more specifically directed to a method for obtaining pulverulent mixtures of amphiphilic lipidic constituents and hydrophobic constituents, the said mixture being used particularly for producing hydrated lipidic lamellar phases.
The hydrated lipidic lamellar phases have been described amply in scientific publications, notably by R. PERRON ("Revue Francaise du Corps Gras", 1980, 27 (4) 173-183). Their formation results from a property which is typical of certain amphiphilic lipids in the presence of an aqueous medium. The amphiphilic lipid molecules are composed of a polar or non-polar hydrophilic portion and of a hydrophobic portion. According to M. M. RIEGER ("Cosmetics and Toiletries", 1981, 96 35-38), one of the conditions for the amphiphilic lipid to be capable of forming lamellar phases rather than only micellar clusters is that the dimensions of its hydrophobic portion be at least as important as that of its hydrophilic portion.
Such lipids are capable of forming bimolecular lipidic layers (lamellae or bilayers), separated from one another by a more or less important amount of water or of aqueous solution which may be reduced to a mere interstitial film. The molecules of said lipids forming the lipidic layers are placed side by side and so oriented that their hydrophobic portion is located inside the layer. The lipidic layers and interstitial aqueous medium as a whole are referred to as a hydrated lipidic lamellar phase.
The liposome is a particular configuration of a lamellar phase appearing in the form of a lipidic spherule composed of one or several concentric bilayers alternating with aqueous compartments. The liposomes are dispersed in an aqueous medium and form a highly hydrated lamellar phase. The conditions under which non-polar amphiphilic lipids can form liposomes or "niosomes" have been set forth by G. VANLERBERGHE et al. ("Colloques Nationaux du CNRS", N.degree. 938, Bordeaux 1978, CNRS Publications, 1979, pages 303-311).
It is also known that liposomes can be obtained by dispersing in an aqueous medium a lamellar phase by various methods, notably by means of ultrasonic waves.
Liposomes have been dealt with in a great number of publications, such as for example in the articles already mentioned and in the following articles: Sessa G. et al., "J. Lipid Res." 1968, 9, 310-318; Bangham A. D. et al., "Meth. Membr. Biol." 1974, 1-68; D. A. Tyrrell et al., "Biochim. Biophys. Acta" 1976, 457, 259-302; Strianse S. J., "Cosmetics & Toiletries" 1978, 93, 37-41; Puisieux F., "Labo-Pharma" 1978, 281, 899-904; Nicolau C. et al., "La Recherche" 1981, 123, 748-749.
The commercial interest of liposomes or of hydrated lamellar phases lies in the fact that substances possessing interesting properties may be incorporated either in the lipidic bilayers in the case of the partially or totally hydrophobic substances, or in solution, in the aqueous medium located between two bilayers, or encapsulated in liposomes in the case of the water-soluble substances.
These products find numerous applications, particularly in the production of foodstuffs, cosmetic or pharmaceutical products.
Widely different substances may be incorporated in the lamellar phases or the liposomes, notably for the purpose of protecting damageable substances from external agents or of improving the penetration of certain substances into the biological tissues. It is also possible to modify the physico-chemical properties of the lamellar phases or the liposomes themselves by incorporating into the lipidic bilayers certain compounds such as sterols, e.g. cholesterol (G. Vanderberghe et al., supra; G. Gregoriadis, "Biochem J", 19, 196, 591) for the purpose, in particular, of controlling the permeability and stability of the lipidic bilayers.
The addition of hydrophobic compounds such as sterols increases the hydrophobic group to hydrophilic group ratio, which complies with the condition of stability of the lamellar phases set forth by M. M. Rieger (supra). Thus, the addition of such hydrophobic compounds makes possible the formation of lamellar phases with amphiphilic lipids which could not form such phases used alone. It is also possible to incorporate into the lamellar phase amphiphilic compounds whose hydrophilic portion is charged positively or negatively (French patent applications No. 78-22985 and No. 78-13632); for example, dicetylphosphate imparts to the liposomes a negative charge, whereas stearylamine provides a positive charge.
A certain number of methods of production of such lamellar phases and of liposomes are already known. One of the methods most currently in use consists, in a first stage, in dissolving the amphiphilic lipid or lipids and possibly the hydrophobic substances or substances in an appropriate volatile solvent. The solution obtained is placed in the flask of a rotary evaporator. After the solvent is evaporated, a film adhering to the flask wall is obtained. Water or, if suitable, the aqueous solution to be encapsulated is introduced into the flask and the entire content is vigorously agitated. There is thus obtained a suspension of liposomes which, if appropriate, may be subjected to ultrasonic homogenization. One of the drawbacks to this method resides in that the film can be obtained only with great difficulty on a commercial scale.
According to another method described notably in French patent application No. 78-22985, the aqueous solution to be encapsulated is dispersed in a solvent which is insoluble or only slightly soluble in water in the presence of a lipid or of an amphiphilic surfactant. This results in the formation of microdrops of the said aqueous solution, which are thereafter emulsified in an aqueous medium in the presence of an excess of a lipid or surfactant which may be identical with or different from the one used previously. The insoluble solvent is eliminated before or after the emulsification, for example by blowing air at the surface of the liquid. This method requires a complete elimination of the solvent used, which involves numerous difficulties, especially on a commercial scale. Moreover, it does not allow for production of only slightly hydrated lamellar phases.
It is also possible to prepare liposomes according to the method of Swiss Pat. No. 623,236 by bringing into contact, on the one hand, at least one liquid amphiliphic lipid dispersible in water, the hydrophilous portion of which is ionic, and on the other hand, the aqueous phase to be encapsulated. The mixture is thereafter subjected to vigorous agitation to obtain a lamellar phase which thereafter may be dispersed in water or an aqueous solution. In this method, to incorporate hydrophobic compounds, it is necessary to use surface-active compounds whose emulsifying property is higher the more marked the hydrophobic character of the compound to be incorporated. Such surface-active compounds are very often incompatible with many applications of the liposomes, e.g. in the field of pharmacy or cosmetics.
According to another method described in French patent application No. 78-13632, a solution is prepared containing at least one amphiphilic lipid, at least one biologically active compound and, if suitable, at least one catalyst. This solution is lyophilized to eliminate the solvent and the mixture of the constituents is obtained in the form of powder which may be stored in sealed containers until it is used to form, in particular, liposomes. This last stage consists in dispersing the powder in an appropriate aqueous medium. This method suffers from the drawbacks involved particularly in the conventional lyophilizing techniques which are relatively complex and expensive and the production rate of which is moreover relatively limited.
The various known methods described above therefore suffer from many drawbacks which, do not allow, in particular, easy or inexpensive production of lamellar phases on a commercial scale. These drawbacks are mainly due to the difficulties of obtaining a thorough mixture of the basic constituents forming the lamellar phases, in other words the amphiphilic lipids and possibly the hydrophobic substances. In order to facilitate the dispersion of the basic constituents of the lamellar phases in an aqueous medium, it is often necessary to use additional products with surface-active properties. Since such products often have undesirable effects, e.g. with respect to the organism, it is necessary to eliminate them completely, which is not always possible even by way of dialysis. "Basic constituents" means the compounds used to form the lipidic layers of the lipidic lamellar phase, namely, at least one amphiphilic lipid or a mixture of one or several amphiphilic lipids and, possibly, one or several partially or totally hydrophobic compounds.